Cell-based assays are increasingly gaining in popularity in the pharmaceutical industry due to their high physiological relevance. Among the advantages of these assays are their ability to predict compound usefulness, evaluate molecular interactions, identify toxicity, distinguish cell type-specific drug effects, and determine drug penetration. Cell-based assays are relevant throughout the drug discovery pipeline because they are capable of providing data from target characterization and validation leading to identification (primary and secondary screening) to terminal stages of toxicology. Current industry trends of performing drug screening with cell context demand easily monitored, non-invasive reporters. Fluorescent proteins fulfill this demand more completely than any other available tools and have demonstrated applicability and versatility as molecular and cellular probes in life sciences and biomedical research. Among patents relating to fluorescent protein technology are U.S. Pat. Nos. 5,491,084; 5,625,048; 5,777,079; 5,804,387; 5,968,738; 5,994,077; 6,027,881; 6,054,321; 6,066,476; 6,077,707; 6,124,128; 6090,919; 6,172,188; 6,146,826; 6,969,597; 7,150,979; 7,157,565; 7,166,444; 7,183,399 and 7,297,782, all incorporated by reference in their entirety.
Requirements for advanced screening assays are driven by the objective of identifying candidate compounds which fail early in the drug discovery pipeline. This fundamental approach increases efficiency, reduces costs, and results in shorter time to market for new drugs. In order to fail compounds early, however, information-rich data for accurate early-stage decision making is required. Such data may be derived by screening compounds in context, that is, by screening in relevant living systems, rather than with classical biochemical assays. To do so, sophisticated imaging platforms, such as high-content screening (HCS) workstations, must often be incorporated. The industrialization of fluorescent microscopy has led to the development of these high-throughput imaging platforms capable of HCS. When coupled with fluorescent protein reporter technology, HCS has provided information-rich drug screens, as well as access to novel types of drug targets.
As industry trends advance toward analysis in living systems (e.g., cells, tissues, and whole organisms), fluorescent proteins, by virtue of their non-invasive, non-destructive properties, are becoming indispensable tools for live-cell analysis. A broad range of fluorescent protein genetic variants are now available, with fluorescence emission profiles spanning nearly the entire visible light spectrum. Mutagenesis efforts in the original jellyfish Aequorea victoria green fluorescent protein have resulted in new fluorescent probes that range in color from blue to yellow and these are some of the most widely used in vivo reporter molecules in biological research today. Longer wavelength fluorescent proteins which emit in the orange and red spectral regions, have been developed from the marine anemone Discosoma striata and reef corals belonging to the class Anthozoa. Other species have also been mined to produce similar proteins having cyan, green, yellow, orange, red, and even far-red fluorescence emission.
Recent emphasis on multi-color imaging in HCS has created renewed demand for easily measured, non-invasive, and non-disruptive cellular and molecular probes. With the increasingly expanded repertoire of fluorescent proteins has come increased demand for complementary reagents, such as organic fluorochrome counter-stains that augment analysis by providing information relating to co-localization of the fluorescent proteins to various organelles and subcellular targets. To date, however, concerted efforts in developing such organic fluorochromes specifically tailored to working in concert with fluorescent proteins, has been limited in scope. The application of fluorescent proteins and organic fluorochromes is not an “either-or” proposition. Each technology has distinct advantages and limitations. These two technologies can be optimized and combined to work in concert, however, in order to maximize the information content obtained from fluorescence microscopy and imaging-based screening approaches. By doing so, achieving rich multi-dimensional physiological information can be obtained.
While suitable for analysis of cell surfaces and permeabilized cells, fluorescently-labeled antibodies have few practical applications for intracellular imaging in living cells. The limited application of fluorescent antibodies stems from due their inherent inability to penetrate their targets. This has given rise to development of cell-permeable small molecule organic fluorochromes, certain ones of which naturally sequester inside-specific organelles, based upon biophysical or biochemical properties favoring that distribution. Acceptable small molecule organic probes for cell imaging and analysis need to be minimally perturbing, versatile, stable, easy-to-use, and easy to detect using non-invasive imaging equipment. A problem with the classical organic probes from histology past is that many of them either require cofactors or fixation and staining, the latter only reporting on the static condition of a dead cell. The required additional steps may be time consuming, expensive and in the case of fixing and staining, lack biological relevance. In the context of the analyses described above, an organic probe must be able to report upon events in living cells and in real time. Simplicity is of key importance, especially in the context of drug screening.
While various organic fluorochromes have been developed in the past for live cell analysis, typically they were not devised with optimization of performance in conjunction with the wide palette of available fluorescent proteins in mind. For instance, several disclosures (U.S. Pat. Nos. 5,338,854; 5,459,268; 5,686,261; 5,869,689; 6,004,536; 6,140,500 and 6,291,203 B1, as well as US Patent Publications Nos. 2005/0054006 and 2007/0111251 A1, references incorporated herein) disclose organic fluorochromes which are described as useful for visualizing membranes, mitochondria, nuclei and/or acidic organelles. Additional examples of various fluorochromes and their application in biological imaging may be found in the published literature (see, for example, Pagano et al, 1989; Pagano et al, 1991; Deng et al, 1995; Poot et al, 1996; Diwu et al, 1999; Rutledge et al, 2000; Lee et al, 2003; Bassøe et al, 2003; Rosania et al, 2003, Li et al 2007; Boldyrev et al, 2007; Nadrigny et al, 2007). These dyes have been created using a number of fluorophores, most commonly dipyrrometheneboron difluoride (BODIPY), cyanine, carbocyanine, styryl and diaminoxanthene core structures. Typical emission maxima for these organic fluorophores span from 430 to 620 nm. Many of the dyes consequently occupy valuable regions of the visible emission spectrum that preclude use of various fluorescent proteins. As such, the use of these dyes limits the overall levels of multiplexing achievable in HCS assays. Additionally, these dyes often display other suboptimal properties, such as a propensity to photo-bleach, metachromasy and even a tendency to photo-convert to different emission maxima upon brief exposure to broad-band illumination. For example, Lysotracker Red DND-99 (Invitrogen, Carlsbad, Calif., a lysosomal stain) which contains a BODIPY fluorophore in the form of a conjugated multi-pyrrole ring structure upon broad-band illumination, induces molecular changes rendering its photochemical properties similar to those of Lysotracker Green. The similarities between the spectra of Lysotracker Green and converted Lysotracker Red suggest that the third pyrrole ring is taken out of conjugation during the photo-conversion process, leading to a shorter wavelength dye emission. Thus, Lysotracker Red staining for epifluorescence or confocal microscopy, in conjunction with visualization of GFP, leads to spurious results due to photo-conversion of the fluorophore (Freundt et al, 2007).
Acridine orange (Sigma-Aldrich, Saint Louis, Mo. and other sources) has also been used extensively as a fluorescent probe of lysosomes and other acidic subcellular compartments. The metachromasy of acridine organe results, however, in the concomitant emission of green and red fluorescence from stained cells and tissue (Nadrigny et al, 2007). Evanescent-field imaging with spectral fluorescence detection, together with fluorescence lifetime imaging microscopy, demonstrate that green fluorescent acridine orange monomers inevitably coexist with red fluorescing acridine orange dimers in labeled cells. The green monomer emission spectrally overlaps with that of GFP and produces a false apparent co-localization on dual-color images. Due to its complicated photochemistry and interaction with cellular constituents, acridine orange is a particularly problematic label for multi-color fluorescence imaging, both for dual-band and spectral detection. Extreme caution is required, therefore, when deriving quantitative co-localization information from images of GFP-tagged proteins in cells co-labeled with acridine orange.
In principle, the styryl dye, FM4-64 (Invitrogen, Carlsbad, Calif.) is useful for studying endocytosis and vesicular recycling because it is reputed to be confined to the luminal layer of endocytic vesicles. This particular dye distributes throughout intracellular membranes and it indiscriminately stains both the endoplasmic reticulum and nuclear envelope (Zal et al, 2006). Though the different pools of dye all emit at roughly 700 nm, a spectral shift in fluorescence excitation maximum is observed, however, because the dye present in endocytic vesicles and the endoplasmic reticulum absorbs at 510 nm, while the dye associated with the nuclear matrix absorbs at 622 nm. While this can be used advantageously in order to selectively image the nuclear membrane, the dual absorption properties can be problematic in certain multi-parametric imaging experiments. The shift in peak of the absorption spectrum is not confined to FM dyes. A similar phenomenon has also been reported for Rhodamine 6G, where the dye's absorbance maximum is red-shifted from 527 to 546 nm in a concentration dependent manner (Johnson et al, 1978). Rhodamine 6G is commonly employed to label leukocytes, especially in vascular injury models.
Fluorescent analogs of ceramide are commonly employed to visualize Golgi bodies in live cells. The fluorescence emission maximum of certain BODIPY-labeled ceramides, such as C5-DMD-Ceramide (a.k.a. C5-BODIPY-Cer, Invitrogen, Carlsbad, Calif.), has been shown to depend strongly upon the molar density of the probe in the membrane, shifting in emission maximum from green (˜515 nm) to red (˜620 nm) with increasing concentration (Pagano et al, 1991). Consequently, in live cells, the Golgi bodies display yellow/orange fluorescence emission (a combination of red and green fluorescence emission), whereas predominantly green fluorescence emission is observed in the endoplasmic reticuli, the nuclear envelope and mitochondria. Due to inherent dual emission characteristics when employing these fluorescent ceramide analogs, co-localization studies with GFP are compromised.
Thus, there is a need for the development of new and better dye molecules with improved photo physical properties, cell-permeability and target specificity to various cell regions. These dye candidates should also have the capability of using in multi color cell analysis for imaging or high-throughput screening.